Insulin-dependent diabetes mellitus (IDDM) is the most common autoimmune disease in humans and its incidence is rising. We have developed molecular tools to study some fundamental aspects of the mechanisms leading to self-reactivity during the first 5 years of this grant. Our effort will be pursued along the same lines. From a structural perspective, the priority will be on achieving the x-ray structureI determination of at least one I-Ag7/TCR complex. Effort will be put into the development of more efficient protein expression systems and the development of single chain FV versions of soluble TCRs. These structures together with measurements of the affinity of self-reactive TCRs for their self-peptide MHC ligands will allow us to understand the role that TCR/MHC interaction may play in the selection and expansion of autoreactive T cells. Structural studies will also be the basis to investigate the role of the anionic P9 pocket of the I-Ag7 molecule on the selection of TCRs with acidic residues within their CDR2a and CDR3b regions, whenever charge complementation is not provided by the peptide. This hypothesis will be tested in the NOD mouse using immunization with variant peptides and TCR sequencing. Recombinant MHC will also be used to produce multimeric reagents able to detect antigen-specific CD4+ T cells in naive NOD using natural peptides (from GAD and insulin) and mimotopes reactive to diabetogenic T cell clones. The initial characterization of the natural BDC2.5-reactive population, that was identified using this approach, will be extended further with quantitative cytokine profiling, single cell TCR analysis and in vivo transfer experiments. A similar strategy will be used to try to identify an equivalent T cell population in HLA-DQ8 transgenic mice and in human using HLA-DQ8 -mimotope multimers. Further improvements of the MHC tetramer technology will be pursued using newer protein engineering techniques to try to obtain a reliable, sensitive technique fit to clinical settings for the detection of pathogenic CD4+ T cells in blood. Finally, we will also use multimerized recombinant MHC molecules to try therapeutic targeting of the BDC2.5+ antigen-specific T cells in the NOD. Efficacy will be appreciated by measuring BDC2.5+ cells using FACS and glucose levels during the course of treatment.